The primary long-term objective of the proposed research is to understand the molecular basis of the hypoxia response pathway. The growth and spread of solid tumors depends on an adequate blood supply, which develops in response to low oxygen conditions. Hepatocellular carcinoma, in addition to being a worldwide cancer problem for which there are few successful treatments, is a cancer that adapts well to hypoxic conditions. Human hepatocellular carcinoma (Hep3B) cells produce erythropoietin (EPO) as well as vascular endothelial growth factor (VEGF) in response to hypoxia. Earlier we demonstrated that protein kinase C alpha activation is necessary: for sustained EPO production over 24 hrs of hypoxia..Subsequent studies done in our laboratory provide evidence for the involvement of the phosphatidylinositol-3 kinase pathway, as well as the mitogen-activated protein kinase (MAPK) in EPO production. Studies of erythropoietin have led to the definition of a widely operative system of gene regulation by oxygen that is dependent on the activation of a transcriptional complex termed hypoxia inducible factor-1(HIF-1). This activation is proposed to be mediated by specific kinase pathways. The specific aims are to: 1) determine the role of PI3K/Akt in EPO gene expression, 2) examine the effects of PI3K on HIF-1 transactivation and, 3) determine the potential cross-talk between P13K and Raf/MAPK/Erk pathways. It is expected that these studies will yield important information regarding the molecular signaling pathways critical for survival of Hep3B cells in hypoxia.